Ultrabithorax Research Articles

Molecular and genetic characterization of Cbx-Basel , a new dominant allele of Ultrabithorax in D. melanogaster.

Dominant gain-of-function alleles for the homeotic gene Ultrabithorax ( Ubx ) have been known for a long time. They are summarized under the name Contrabithorax ( Cbx ). Such alleles are rather easy to spot because the morphology of the conspicuous dorsal wing appendage is often dramatically changed. The majority of these alleles is associated with chromosomal rearrangements that alter the genetic landscape of the Ultrabithorax locus. Thereby, UBX protein is ectopically expressed in the wing primordium where it is normally absent. Since Ubx specifies haltere identity, wing cells expressing UBX are determined to become haltere cells. However, apart from the prototypic allele Cbx-1 , information on the molecular details of Contrabithorax alleles is scarce. Here, we present a rather detailed account on a novel Cbx-1-like allele called Cbx-Basel . The results of our study corroborate the model that has been postulated for the Cbx-1 wing phenotype.

LncRNA read-through regulates the BX-C insulator Fub-1.

hough long non-coding RNAs (lncRNAs) represent a substantial fraction of the Pol II transcripts in multicellular animals, only a few have known functions. Here we report that the blocking activity of the Bithorax complex (BX-C) Fub-1 boundary is segmentally regulated by its own lncRNA. The Fub-1 boundary is located between the Ultrabithorax (Ubx) gene and the bxd/pbx regulatory domain, which is responsible for regulating Ubx expression in parasegment PS6/segment A1. Fub-1 consists of two hypersensitive sites, HS1 and HS2. HS1 is an insulator while HS2 functions primarily as a lncRNA promoter. To activate Ubx expression in PS6/A1 enhancers in the bxd/pbx domain must be able to bypass Fub-1 blocking activity. We show that expression of the Fub-1 lncRNAs in PS6/A1 from the HS2 promoter inactivates Fub-1 insulating activity. Inactivation is due to readthrough as the HS2 promoter must be directed towards HS1 to disrupt blocking.

A novel post-developmental role of the Hox genes underlies normal adult behavior

The molecular mechanisms underlying the stability of mature neurons and neural circuits are poorly understood. Here we explore this problem and discover that the Hox genes are a component of the genetic program that maintains normal neural function in adult Drosophila. We show that post-developmental downregulation of the Hox gene Ultrabithorax (Ubx) in adult neurons leads to substantial anomalies in flight. Mapping the cellular basis of these effects reveals that Ubx is required within a subset of dopaminergic neurons, and cell circuitry analyses in combination with optogenetics allow us to link these dopaminergic neurons to flight control. Functional imaging experiments show that Ubx is necessary for normal dopaminergic activity, and neuron-specific RNA-sequencing defines two previously uncharacterized ion channel-encoding genes as potential mediators of Ubx behavioral roles. Our study thus reveals a novel role of the Hox system in controlling adult behavior and neural function.Based on the broad evolutionary conservation of the Hox system across distantly related animal phyla, we predict that the Hox genes might play neurophysiological roles in adult forms of other species, including humans.

Ultrabithorax modifies a regulatory network of genes essential for butterfly eyespot development in a wing sector-specific manner.

Nymphalid butterfly species often have a different number of eyespots in forewings and hindwings, but how the hindwing identity gene Ultrabithorax (Ubx) drives this asymmetry is not fully understood. We examined a three-gene regulatory network for eyespot development in the hindwings of Bicyclus anynana butterflies and compared it with the same network previously described for forewings. We also examined how Ubx interacts with each of these three eyespot-essential genes. We found similar genetic interactions between the three genes in fore- and hindwings, but we discovered three regulatory differences: Antennapedia (Antp) merely enhances spalt (sal) expression in the eyespot foci in hindwings, but is not essential for sal activation, as in forewings; Ubx upregulates Antp in all hindwing eyespot foci but represses Antp outside these wing regions; and Ubx regulates sal in a wing sector-specific manner, i.e. it activates sal expression only in the sectors that have hindwing-specific eyespots. We propose a model for how the regulatory connections between these four genes evolved to produce wing- and sector-specific variation in eyespot number.

Flight or protection: the genes Ultrabithorax and apterous in the determination of membranous and sclerotized wings in insects.

Present-day pterygote insects have two pairs of wings, one in the mesothorax (T2), the other in the metathorax (T3), and both have diverged in structure and function in different groups. Studies in endopterygote and paraneopteran species have shown that the gene Ultrabithorax (Ubx) specifies the identity and wing structure in T3, whereas the gene apterous (ap) significantly contributes to forming modified T2 wings. We wondered whether these Ubx and ap mechanisms operate in the lineage of polyneopterans. To explore this possibility, we used the cockroach Blattella germanica (Polyneoptera and Blattodea), in which the T2 wings are sclerotized (tegmina), whereas those of the T3 are membranous. We found that Ubx determines the structure of T3 and the membranous wing, while ap significantly contributes to form the sclerotized T2 tegmina. These results along with the studies carried out on the beetle Tribolium castaneum by Tomoyasu and collaborators suggest that ap plays an important role in the sclerotization and melanization of the T2 wings in neopteran groups that have sclerotized forewings. In turn, the sclerotizing properties of ap demonstrated in beetles and cockroaches suggest that the origin of this function goes back to the emergence of Neoptera, in the mid Devonian.

Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins

SummaryUsing the Drosophila melanogaster Hox gene Ultrabithorax (Ubx) as an example, we demonstrate the use of three heterologous DNA-binding protein systems—LacI/LacO, ParB1/ParS1, and ParB2/ParS2—to label genomic loci in imaginal discs with the insertion of a small DNA tag. We compare each system, considering the impact of labeling in genomic regions (1) inside versus outside of a transcribed gene body and (2) with varying chromatin accessibility. We demonstrate the value of this system by interrogating the relationship between gene expression level and enhancer-promoter distance, as well as inter-allelic distance at the Ubx locus. We find that the distance between an essential intronic cis-regulatory element, anterobithorax (abx), and the promoter does not vary with expression level. In contrast, inter-allelic distance correlates with Ubx expression level.

Wing hearts in four-winged Ultrabithorax-mutant flies-the role of Hox genes in wing heart specification.

Wings are probably the most advanced evolutionary novelty in insects. In the fruit fly Drosophila melanogaster, proper development of wings requires the activity of so-called wing hearts located in the scutellum of the thorax. Immediately after the imaginal ecdysis, these accessory circulatory organs remove hemolymph and apoptotic epidermal cells from the premature wings through their pumping action. This clearing process is essential for the formation of functional wing blades. Mutant flies that lack intact wing hearts are flightless and display malformed wings. The embryonic wing heart progenitors originate from two adjacent parasegments corresponding to the later second and third thoracic segments. However, adult dipterian flies harbor only one pair of wings and only one pair of associated wing hearts in the second thoracic segment. Here we show that the specification of WHPs depends on the regulatory activity of the Hox gene Ultrabithorax. Furthermore, we analyzed the development of wing hearts in the famous four-winged Ultrabithorax (Ubx) mutant, which was first discovered by Ed Lewis in the 1970s. In these flies, the third thoracic segment is homeotically transformed into a second thoracic segment resulting in a second pair of wings instead of the club-shaped halteres. We show that a second pair of functional wing hearts is formed in the transformed third thoracic segment and that all wing hearts originate from the wild-type population of wing heart progenitor cells.

Integrative Role of 14-3-3ε in Sleep Regulation

Sleep is a crucial factor for health and survival in all animals. In this study, we found by proteomic analysis that some cancer related proteins were impacted by the circadian clock. The 14-3-3ε protein, expression of which is activated by the circadian transcription factor Clock, regulates adult sleep of Drosophila independent of circadian rhythm. Detailed analysis of the sleep regulatory mechanism shows that 14-3-3ε directly targets the Ultrabithorax (Ubx) gene to activate transcription of the pigment dispersing factor (PDF). The dopamine receptor (Dop1R1) and the octopamine receptor (Oamb), are also involved in the 14-3-3ε pathway, which in 14-3-3ε mutant flies causes increases in the dopR1 and OAMB, while downregulation of the DopR1 and Oamb can restore the sleep phenotype caused by the 14-3-3ε mutation. In conclusion, 14-3-3ε is necessary for sleep regulation in Drosophila.

Developmental Robustness: The Haltere Case in Drosophila.

Developmental processes have to be robust but also flexible enough to respond to genetic and environmental variations. Different mechanisms have been described to explain the apparent antagonistic nature of developmental robustness and plasticity. Here, we present a “self-sufficient” molecular model to explain the development of a particular flight organ that is under the control of the Hox gene Ultrabithorax (Ubx) in the fruit fly Drosophila melanogaster. Our model is based on a candidate RNAi screen and additional genetic analyses that all converge to an autonomous and cofactor-independent mode of action for Ubx. We postulate that this self-sufficient molecular mechanism is possible due to an unusually high expression level of the Hox protein. We propose that high dosage could constitute a so far poorly investigated molecular strategy for allowing Hox proteins to both innovate and stabilize new forms during evolution.

An InR/mir‐9a/NlUbx regulatory cascade regulates wing diphenism in brown planthoppers

Wing polymorphism significantly contributes to the ecological success of some insect species. For example, the brown planthopper (BPH) Nilaparvata lugens, which is one of the most destructive rice pests in Asia, can develop into either highly mobile long-winged or highly fecund short-winged adult morphs. A recent study reported a highly provocative result that the Hox gene Ultrabithorax (Ubx) is expressed in BPH forewings and showed that this wing development gene is differentially expressed in nymphs that develop into long-winged versus short-winged morphs. Here, we found that Ubx may be a mir-9a target, and used dual luciferase reporter assays and injected micro RNA (miRNA) mimics and inhibitors to confirm the interactions between mir-9a and NlUbx. We measured the mir-9a and NlUbx expression profiles in nymphs and found that the expression of these two biomolecules was negatively correlated. By rearing BPH nymphs on host rice plants with different nutritional status, we were able to characterize a regulatory cascade between insulin receptor genes, mir-9a, and NlUbx that regulate wing length in BPHs. When host quality was low, NlInR1 expression in the nymph terga increased and NlInR2 expression decreased; this led to a higher mir-9a level, which in turn reduced the NlUbx transcript level and ultimately resulted in longer wing lengths. Beyond extending our understanding of the interplay between host plant status and genetic events that modulate polymorphism, we demonstrated both the upstream signal and miRNA-based regulatory mechanism that control Ubx expression in BPH forewings.

Control of tissue morphogenesis by the HOX gene Ultrabithorax.

ABSTRACTMutations in the Ultrabithorax (Ubx) gene cause homeotic transformation of the normally two-winged Drosophila into a four-winged mutant fly. Ubx encodes a HOX family transcription factor that specifies segment identity, including transformation of the second set of wings into rudimentary halteres. Ubx is known to control the expression of many genes that regulate tissue growth and patterning, but how it regulates tissue morphogenesis to reshape the wing into a haltere is still unclear. Here, we show that Ubx acts by repressing the expression of two genes in the haltere, Stubble and Notopleural, both of which encode transmembrane proteases that remodel the apical extracellular matrix to promote wing morphogenesis. In addition, Ubx induces expression of the Tissue inhibitor of metalloproteases in the haltere, which prevents the basal extracellular matrix remodelling necessary for wing morphogenesis. Our results provide a long-awaited explanation for how Ubx controls morphogenetic transformation.

Functional analysis of Ultrabithorax in the wing-dimorphic planthopper Nilaparvata lugens (Stål, 1854) (Hemiptera: Delphacidae)

The homeotic complex (Hox) gene Ultrabithorax (Ubx) plays pivotal roles in modifying specific morphological differences among the second (T2), the third thoracic (T3), and the first abdomen (A1) segment in several insects. Whether Ubx regulates wing dimorphism and other morphological traits in the delphacid family (order Hemiptera) remains elusive. In this study, we cloned a full-length Ubx ortholog (NlUbx) from the wing-dimorphic planthopper Nilaparvata lugens, and identified two NlUbx isoforms. RNA-interference (RNAi)-mediated silencing of NlUbx in short-winged BPH nymphs significantly induced the development of wing-like appendages from T3 wingbuds, and this effect is likely mediated by the insulin/insulin-like signaling pathway. RNAi knockdown of NlUbx in long-winged BPH nymphs led to a transformation from hindwings to forewings. Additionally, silencing of NlUbx not only dramatically changed the T3 morphology, but also led to jumping defect of T3 legs. First-instar nymphs derived from parental RNAi had an additional leg-like appendages on A1. These results suggest that Ubx plays a role in determining some morphological traits in delphacid planthoppers, and thus help in understanding evolution of morphological characteristics in arthropods.

A Single MicroRNA-Hox Gene Module Controls Equivalent Movements in Biomechanically Distinct Forms of Drosophila

SummaryMovement is the main output of the nervous system. It emerges during development to become a highly coordinated physiological process essential to survival and adaptation of the organism to the environment. Similar movements can be observed in morphologically distinct developmental stages of an organism, but it is currently unclear whether or not these movements have a common molecular cellular basis. Here we explore this problem in Drosophila, focusing on the roles played by the microRNA (miRNA) locus miR-iab4/8, which we previously showed to be essential for the normal corrective response displayed by the fruit fly larva when turned upside down (self-righting). Our study shows that miR-iab4 is required for normal self-righting across all three Drosophila larval stages. Unexpectedly, we also discover that this miRNA is essential for normal self-righting behavior in the adult fly, an organism with different morphology, neural constitution, and biomechanics. Through the combination of gene expression, optical imaging, and quantitative behavioral approaches, we provide evidence that miR-iab4 exerts its effects on adult self-righting behavior in part through repression of the Hox gene Ultrabithorax (Ubx) in a specific set of adult motor neurons, the NB2-3/lin15 neurons. Our results show that miRNA controls the function, rather than the morphology, of these neurons and demonstrate that post-developmental changes in Hox gene expression can modulate behavior in the adult. Our work reveals that a common miRNA-Hox genetic module can be re-deployed in different neurons to control functionally equivalent movements in biomechanically distinct organisms and describes a novel post-developmental role of the Hox genes in adult neural function.

Rapid and efficient purification of Drosophila homeodomain transcription factors for biophysical characterization

Homeodomain transcription factors (HD TFs) are a large class of evolutionarily conserved DNA binding proteins that contain a basic 60-amino acid region required for binding to specific DNA sites. In Drosophila melanogaster, many of these HD TFs are expressed in the early embryo and control transcription of target genes in development through their interaction with cis-regulatory modules. Previous studies where some of the Drosophila HD TFs were purified required the use of strong denaturants (i.e. 6 M urea) and multiple chromatography columns, making the downstream biochemical examination of the isolated protein difficult. To circumvent these obstacles, we have developed a streamlined expression and purification protocol to produce large yields of Drosophila HD TFs. Using the HD TFs FUSHI-TARAZU (FTZ), ANTENNAPEDIA (ANTP), ABDOMINAL-A (ABD-A), ABDOMINAL-B (ABD-B), and ULTRABITHORAX (UBX) as examples, we demonstrate that our 3-day protocol involving the overexpression of His6-SUMO fusion constructs in E. coli followed by a Ni2+-IMAC, SUMO-tag cleavage with the SUMO protease Ulp1, and a heparin column purification produces pure, soluble protein in biological buffers around pH 7 in the absence of denaturants. Electrophoretic mobility shift assays (EMSA) confirm that the purified HD proteins are functional and nuclear magnetic resonance (NMR) spectra confirm that the purified HDs are well-folded. These purified HD TFs can be used in future biophysical experiments to structurally and biochemically characterize how and why these HD TFs bind to different DNA sequences and further probe how nucleotide differences contribute to TF-DNA specificity in the HD family.

Hox genes mediate the escalation of sexually antagonistic traits in water striders.

Sexual conflict occurs when traits favoured in one sex impose fitness costs on the other sex. In the case of sexual conflict over mating rate, the sexes often undergo antagonistic coevolution and escalation of traits that enhance females' resistance to superfluous mating and traits that increase males' persistence. How this escalation in sexually antagonistic traits is established during ontogeny remains unclear. In the water strider Rhagovelia antilleana, male persistence traits consist of sex combs on the forelegs and multiple rows of spines and a thick femur in the rear legs. Female resistance traits consist of a prominent spike-like projection of the pronotum. RNAi knockdown against the Hox gene Sex Combs Reduced resulted in the reduction in both the sex comb in males and the pronotum projection in females. RNAi against the Hox gene Ultrabithorax resulted in the complete loss or reduction of all persistence traits in male rear legs. These results demonstrate that Hox genes can be involved in intra- and inter-locus sexual conflict and mediate escalation of sexually antagonistic traits.

MicroRNA-dependent regulation of Hox gene expression sculpts fine-grain morphological patterns in a Drosophila appendage.

ABSTRACTDisruptions of normal Hox gene expression can lead to severe morphological defects, revealing a link between the regulation of Hox expression and pattern formation. Here, we explore these links, focusing on the impact of microRNA regulation on the expression of the Drosophila Hox gene Ultrabithorax (Ubx) during haltere development. Through a combination of bioinformatic and transcriptomic analyses, we identify the miR-310/313 cluster (miR-310C) as a candidate regulator of Ubx. Several experiments confirm this. First, miR-310C and Ubx protein show complementary expression patterns in haltere imaginal discs; second, artificial activation of miR-310C expression in haltere discs leads to Ubx-like phenotypes. Third, expression of a fluorescent reporter bearing Ubx 3′UTR sequences is reduced when co-expressed with miR-310C. Fourth, deletion of miR-310C leads to Ubx upregulation and changes the array of mechanosensory sensilla at the base of the haltere. Fifth, an artificial increase of Ubx levels within the miR-310C expression domain phenocopies the mechanosensory defects observed in miR-310C mutants. We propose that miR-310C-mediated repression delimits Ubx fine-grain expression, contributing to the sculpting of complex morphologies in the Drosophila haltere with implications for flight control. Our work reveals a novel role of microRNA regulation in the control of Hox gene expression with impact on morphology.

The Drosophila Hox gene Ultrabithorax controls appendage shape by regulating extracellular matrix dynamics.

Although the specific form of an organ is frequently important for its function, the mechanisms underlying organ shape are largely unknown. In Drosophila, the wings and halteres, homologous appendages of the second and third thoracic segments, respectively, bear different forms: wings are flat, whereas halteres are globular, and yet both characteristic shapes are essential for a normal flight. The Hox gene Ultrabithorax (Ubx) governs the difference between wing and haltere development, but how Ubx function in the appendages prevents or allows flat or globular shapes is unknown. Here, we show that Ubx downregulates Matrix metalloproteinase 1 (Mmp1) expression in the haltere pouch at early pupal stage, which in turn prevents the rapid clearance of Collagen IV compared with the wing disc. This difference is instrumental in determining cell shape changes, expansion of the disc and apposition of dorsal and ventral layers, all of these phenotypic traits being characteristic of wing pouch development. Our results suggest that Ubx regulates organ shape by controlling Mmp1 expression, and the extent and timing of extracellular matrix degradation.

Specific expression and function of the Six3 optix in Drosophila serially homologous organs.

ABSTRACTOrgan size and pattern results from the integration of two positional information systems. One global information system, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. However, the mechanisms of the Hox-signal integration in this well-studied system are incomplete. Here, we have investigated this issue by studying the expression and function of the Six3 transcription factor optix during Drosophila wing and haltere development. We find that in both organs, Dpp defines the expression domain of optix through repression, and that the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. We show that optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. In Drosophila, optix is necessary for the growth of wing and haltere. In the wing, optix is required for the growth of the most anterior/proximal region (the ‘marginal cell’) and for the correct formation of sensory structures along the proximal anterior wing margin; the halteres of optix mutants are also significantly reduced. In addition, in the haltere, optix is necessary for the suppression of sensory bristles.

Non-specificity of transcription factor function in Drosophila melanogaster.

A major problem in developmental genetics is how HOX transcription factors, like Proboscipedia (PB) and Ultrabithorax (UBX), regulate distinct programs of gene expression to result in a proboscis versus a haltere, respectively, when the DNA-binding homeodomain (HD) of HOX transcription factors recognizes similar DNA-binding sequences. Indeed, the lack of DNA-binding specificity is a problem for all transcription factors (TFs), as the DNA-binding domains generally recognize small targets of five to six bases in length. Although not the initial intent of the study, I found extensive non-specificity of TF function. Multiple TFs including HOX and HD-containing and non-HD-containing TFs induced both wingless and eyeless phenotypes. The TFs Labial (LAB), Deformed (DFD), Fushi tarazu (FTZ), and Squeeze (SQZ) induced ectopic larval thoracic (T) 1 beard formation in T2 and T3. The TF Doublesex male (DSXM) rescued the reduced maxillary palp pb phenotype. These examples of non-specificity of TF function across classes of TFs, combined with previous observations, compromise the implicit, initial assumption often made that an intrinsic mechanism of TF specificity is important for function. Interestingly, the functional complementation of the pb phenotype may suggest a larger role for regulation of expression of TFs in restriction of function as opposed to an intrinsic specificity of TF function. These observations have major ramifications for analysis of functional conservation in evolution and development.

Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity.

Successful design of tissue engineering scaffolds must include the ability to stimulate vascular development by incorporating angiogenic growth factors. Current approaches can allow diffusion of growth factors, incorporate active factors randomly, or can leave residual toxins. We addressed these problems by genetically fusing the gene encoding Vascular Endothelial Growth Factor (VEGF) with the Ultrabithorax (Ubx) gene to produce fusion proteins capable of self-assembly into materials. We demonstrate that VEGF-Ubx materials enhance human endothelial cell migration, prolong cell survival, and dose-dependently activate the VEGF signaling pathway. VEGF-Ubx fibers attract outgrowing sprouts in an aortic ring assay and induce vessel formation in a chicken embryo chorioallantoic membrane (CAM) assay. Collectively, these results demonstrate that the activity of VEGF remains intact in Ubx materials. This approach could provide an inexpensive and facile mechanism to stimulate and pattern angiogenesis.